Tuning of release kinetics in hydrogels

ABSTRACT

The present disclosure is directed to methods for tuning the release profile of a biologic disposed in a hydrogel. Parameters that can be used for the tuning include a molar ratio of a nucleophilic group to an electrophilic group, the number of the nucleophilic groups in the first precursor, the number of the electrophilic groups in the second precursor, the molecular weight of the first precursor, the molecular weight of the second precursor, a weight ratio of the biologic and excipient to the hydrogel, a weight percentage of the biologic in a solid state formulation, and a ratio of surface area to volume of the hydrogel. The methods described herein allow the formation of hydrogel with different release profiles suitable for different therapeutic applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of International Patent ApplicationNo. PCT/US2019/040940, filed on Jul. 9, 2019, which claims the benefitof priority to U.S. Provisional Application No. 62/695,472, filed onJul. 9, 2018, the contents of each of which are hereby incorporated byreference in their entireties.

FIELD OF THE INVENTION

The present invention relates to hydrogels loaded with biologics andmethods of tuning the release kinetics in hydrogels.

BACKGROUND OF THE INVENTION

Therapeutic agents require a means of delivery to be effective. Drugdelivery relates to administering a pharmaceutical compound to achieve atherapeutic effect in humans or animals. Delivery mechanisms thatprovide release of an agent over time are useful. Drug deliverytechnologies can help to modify a drug release profile, absorption,distribution or drug elimination for the benefit of improving productefficacy and safety, as well as patient convenience and compliance.

SUMMARY OF THE INVENTION

One aspect of the present disclosure relates to a method of producing ahydrogel with a desired release profile for a biologic disposed therein,wherein the biologic is in a solid state formulation prior to beingdisposed in the hydrogel, the method comprising: (i) predetermining atleast one of the following parameters: (a) the number of nucleophilicgroups in a first precursor; (b) the number of electrophilic groups in asecond precursor; (c) the molecular weight of the first precursor; (d)the molecular weight of the second precursor; (e) a weight ratio of thebiologic and excipient to the hydrogel; (f) a weight percentage of thebiologic in the solid state formulation; and (g) a ratio of surface areato volume of the hydrogel; (ii) determining a molar ratio of thenucleophilic group to the electrophilic group alone, or in combinationwith any one or more of the above parameters that is not predetermined,until the desired release profile is achieved; and (iii) crosslinkingthe first precursor and the second precursor at the determined molarratio around the solid state formulation under anhydrous conditions.

In some embodiments, the molar ratio of the nucleophilic group to theelectrophilic group is greater than 1.

In some embodiments, the molar ratio of the nucleophilic group to theelectrophilic group is less than 1.

In some embodiments, the molar ratio of the nucleophilic group to theelectrophilic group is in the range of about 1.1 to about 2.

In some embodiments, the biologic is a recombinant protein, such as anantibody and a Trap protein (a fusion protein with decoy receptordomains).

In some embodiments, the electrophilic group comprises succinimide,succinimide ester, n-hydroxysuccinimide, maleimide, succinate,nitrophenyl carbonate, aldehyde, vinylsulfone, azide, hydrazide,isocyanate, diisocyanate, tosyl, tresyl, or carbonyldiimidazole.

In some embodiments, the nucleophilic group comprises a primary amine ora primary thiol.

In some embodiments, the number of the nucleophilic groups in the firstprecursor is in the range of about 2 to about 10, such as 4 or 8.

In some embodiments, the number of the electrophilic groups in thesecond precursor is in the range of about 2 to about 10, such as 4 or 8.

In some embodiments, the first precursor comprises (aminopropyl)_(m)polyoxyethylene, wherein m is in the range of about 2 to about 10.

In some embodiments, the molecular weight of the first precursor is inthe range of about 1 kDa to about 100 kDa.

In some embodiments, the second precursor comprises(succinimidyloxyglutaryl)_(n) polyoxyethylene, wherein n is in the rangeof about 2 to about 10.

In some embodiments, the molecular weight of the second precursor is inthe range of about 1 kDa to about 100 kDa.

In some embodiments, the weight ratio of the biologic to the hydrogel isbetween about 10% to about 90%. In some embodiments, the weightpercentage of the biologic in the solid state formulation is betweenabout 30% to about 95%.

In some embodiments, the method further comprises producing the solidstate formulation by spray drying, milling, crystallization,precipitation, spray freezing, super critical fluid drying,electrospraying, or microtemplating.

In some embodiments, the method of producing the solid state formulationis spray drying.

In some embodiments, the solid state formulation comprises particles of≤20 μm in diameter.

In some embodiments, the desired release profile comprises a releaseperiod of about two months to six months for at least 90% biologicrelease.

In some embodiments, the desired release profile comprises a releaseperiod of about one week to two months for at least 90% biologicrelease.

In some embodiments, the desired release profile exhibits near-linearrelease over at least one week.

In some embodiments, the desired release profile comprises adelayed-release portion, a sigmoidal shape, a linear portion, anear-linear portion, a logarithmic portion, an exponential portion, or acombination thereof.

In some embodiments, the crosslinking occurs in the presence of anorganic solvent that is anhydrous and hydrophobic.

In some embodiments, the organic solvent is methylene chloride, ethylacetate, dimethyl carbonate, chloroform, or a combination thereof.

In some embodiments, the determining step is performed with a predictivemodel.

In some embodiments, the molar ratio has a continuous effect on releaseprofile in the predictive model. In some embodiments, the release periodin the release profile can be adjusted at a rate of about −41 days permolar ratio change when the molar ratio is greater than 1, e.g., in therange of about 1.3 to about 1.8. In some embodiments, the release periodin the release profile can be adjusted at a rate of about 103 days permolar ratio change when the molar ratio is less than 1, e.g., in therange of about 0.77 to about 0.56.

In some embodiments, the molecular weights of the first and secondprecursors have a non-continuous effect on release profile in thepredictive model.

In another aspect, the present disclosure relates to a method ofproducing a hydrogel having a biologic disposed therein, wherein thebiologic is in the solid state formulation prior to being disposed inthe hydrogel, and wherein the hydrogel is characterized by a desiredrelease period of about one week to about six months for at least 90%biologic release, the method comprising: (i) selecting a first precursorthat comprises two or more nucleophilic groups, wherein the firstprecursor has a molecular weight in the range of about 1 kDa to about100 kDa; (ii) selecting a second precursor that comprises two or moreelectrophilic groups, wherein the second precursor has a molecularweight in the range of about 1 kDa to about 100 kDa; (iii) determiningat least one of the following parameters alone, or in combination, untilthe desired release period is achieved: (a) a molar ratio of thenucleophilic group to the electrophilic group; (b) a weight ratio of thebiologic and excipient to the hydrogel; (c) a weight percentage of thebiologic in a solid state formulation; and (d) a ratio of surface areato volume of the hydrogel; and (iv) crosslinking the first precursor andthe second precursor at the determined molar ratio around the solidstate formulation under anhydrous conditions.

In some embodiments, the molar ratio of the nucleophilic group to theelectrophilic group is greater than 1.

In some embodiments, the molar ratio of the nucleophilic group to theelectrophilic group is less than 1.

In some embodiments, the molar ratio of the nucleophilic group to theelectrophilic group is in the range of about 1.1 to about 2.

In some embodiments, the biologic is a recombinant protein, such as anantibody and a Trap protein.

In some embodiments, the electrophilic group comprises succinimide,succinimide ester, n-hydroxysuccinimide, maleimide, succinate,nitrophenyl carbonate, aldehyde, vinylsulfone, azide, hydrazide,isocyanate, diisocyanate, tosyl, tresyl, or carbonyldiimidazole.

In some embodiments, the nucleophilic group comprises a primary amine ora primary thiol.

In some embodiments, the first precursor comprises about 2 to about 10nucleophilic groups, such as 4 or 8 nucleophilic groups.

In some embodiments, the second precursor comprises about 2 to about 10electrophilic groups, such as 4 or 8 electrophilic groups.

In some embodiments, the first precursor comprises (aminopropyl)_(m)polyoxyethylene, wherein m is in the range of about 2 to about 10.

In some embodiments, the second precursor comprises(succinimidyloxyglutaryl)_(n) polyoxyethylene, wherein n is in the rangeof about 2 to about 10.

In some embodiments, the weight ratio of the biologic to the hydrogel isbetween about 10% to about 90%. In some embodiments, the weightpercentage of the biologic in the solid state formulation is betweenabout 30% to about 95%.

In some embodiments, the method further comprises producing the solidstate formulation by a method selected from the group consisting ofspray drying, milling, crystallization, precipitation, spray freezing,super critical fluid drying, electrospraying, and microtemplating.

In some embodiments, the method of producing the solid state formulationis spray drying.

In some embodiments, the solid state formulation comprises particles of≤20 μm in diameter.

In some embodiments, the desired release period comprises a releaseperiod of about two months to six months for at least 90% biologicrelease.

In some embodiments, the desired release period comprises a releaseperiod of about one week to two months for at least 90% biologicrelease.

In some embodiments, during the desired release period, the hydrogelproduces near-linear release of the biologic.

In some embodiments, the desired release period comprises adelayed-release portion, a sigmoidal shape, a linear portion, anear-linear portion, a logarithmic portion, an exponential portion, or acombination thereof.

In some embodiments, the crosslinking occurs in the presence of anorganic solvent that is anhydrous and hydrophobic, such as methylenechloride, ethyl acetate, dimethyl carbonate, chloroform, or acombination thereof.

In some embodiments, the determining step is performed with a predictivemodel.

In some embodiments, the molar ratio has a continuous effect on releaseperiod in the predictive model. In some embodiments, the release periodin the release profile can be adjusted at a rate of about −41 days permolar ratio change when the molar ratio is greater than 1, e.g., in therange of about 1.3 to about 1.8. In some embodiments, the release periodin the release profile can be adjusted at a rate of about 103 days permolar ratio change when the molar ratio is less than 1, e.g., in therange of about 0.77 to about 0.56.

In some embodiments, the molecular weights of the first and secondprecursors have a non-continuous effect on release period in thepredictive model.

In another aspect, the present disclosure relates to a hydrogelcomprising a biologic produced by the methods described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic explaining the characteristic sigmoidal releaseprofile of a monoclonal antibody (mAb) from a hydrogel.

FIG. 2 is a graph showing the impact of molar ratio on release profile.Experimental release profiles are fit with a Logistic 5 parameter (L5P)curve. All protein loaded cross-linked PEG (XPEG) hydrogels shown inthis figure are mAb IgG1-XPEG with 81.9% w/w protein content in thespray dried formulation, 40 kDa PEG-NHS HGEO and 15 kDa PEG-NH HGEO PEGcomponents, with molar ratios of 1.1, 1.3, and 1.8 and solid loadings of60%, 60%, and 70% respectively.

FIG. 3 is a graph showing that molecule has minimal impact on releaseprofile. Experimental release profiles are fit with an L5P curve. Allprotein loaded hydrogels shown in this figure contain spray driedformulations with 90% w/w protein content, 40 kDa PEG-NHS HGEO and 15kDa PEG-NH HGEO PEG components, 60% w/w solid loading, molar ratiosaround 1.8-1.9, and surface area to volume ratios around 3 mm⁻¹-4 mm⁻¹.Release from mAb IgG4-XPEG is shown in open circles, and release frommAb IgG1-XPEG is shown in closed circles. As both mAbs are similar insize, the primary property impacting diffusion through a porous matrix,it is expected that release kinetics from the hydrogel will be moleculeindependent.

FIG. 4 is a graph showing the impact of protein content in the spraydried formulation on release profile. All protein loaded hydrogels shownin this figure contains mAb IgG1, 60% loading, 1.5 molar ratio, and 40kDa PEG-NHS HGEO and 15 kDa PEG-NH HGEO PEG components with 90% w/wprotein content and 4 mm⁻¹ surface area to volume ratio (squares) or 50%w/w protein content and 35 mm⁻¹ surface area to volume ratio (diamonds).Data have not been normalized for surface area to volume ratio, howeverby comparing this data set with FIG. 6, it is clear that the observeddifference in release profile is not attributed to the surface area tovolume ratio alone as the trends are opposite. Thus, the observeddifference in this data set is attributed to the protein content in thespray dried formulation and/or a combined effect of surface area tovolume ratio and the protein content in the spray dried formulation.

FIG. 5 is a graph showing the impact of solid loading on releaseprofile. All protein loaded hydrogels shown in this figure contains 1.5molar ratio, 40 kDa PEG-NHS HGEO and 15 kDa PEG-NH HGEO PEG components,and 50% protein content with mAb IgG4, 30% solid loading, and 79 mm⁻¹surface area to volume ratio (squares) or mAb IgG1, 60% solid loading,and 35 mm⁻¹ surface area to volume ratio (diamonds). FIG. 3 illustratesthat molecule has minimal impact on release profile. FIGS. 5 and 6illustrate that increasing surface area to volume ratio has a directeffect on the initial burst release. Therefore, the difference indissolution phase of the release profiles for the data sets shown inthis figure is attributed to the impact of solid loading. The overalleffect on the release profile is a result of the combined effect ofsolid loading and surface area to volume ratio.

FIG. 6 is a graph showing the impact of form factor as described bysurface area to volume ratio on release profile, in particular theinitial burst release. All protein loaded hydrogels shown in this figurecontain 40 kDa PEG-NHS HGEO and 15 kDa PEG-NH HGEO PEG components, mAbIgG1, 80% protein content, 80% solid loading, and 1.7 molar ratio withlow surface area to volume ratio in slab form, 12 mm⁻¹ (squares), orhigh surface area to volume ratio in the form of microparticles, >30mm⁻¹ (diamonds).

FIG. 7 is a graph showing how multiple factors can be adjusted togetherto tune release profiles to achieve near linear release for 1-3 months.All protein loaded hydrogels shown in this figure contain 40 kDa PEG-NHSHGEO and 15 kDa PEG-NH HGEO PEG components, mAb IgG1, 82% proteincontent in slab form with 1.8 molar ratio and 60% solid loading(diamonds), 1.7 molar ratio and 80% solid loading (squares), or 1.5molar ratio and 90% solid loading (triangles).

FIG. 8 is a graph showing near linear release for five different proteinloaded hydrogel formulations with four different mAbs and one Trapprotein (i.e., a fusion protein with decoy receptor domains). Allprotein-loaded hydrogel formulations shown in this figure contain 40 kDaPEG-NHS HGEO and 15 kDa PEG-NH HGEO PEG components.

FIG. 9 is a graph showing how, in microparticle format with high surfacearea to volume ratio, release profiles can be tuned to be more linearwith longer duration through adjusting a combination of factorsincluding molar ratio, protein content, and solid loading. All proteinloaded hydrogel formulations shown in this figure contain 40 kDa PEG-NHSHGEO and 15 kDa PEG-NH HGEO PEG components and mAb IgG1 and are producedas microparticles.

FIG. 10 shows model analysis for the study performed in Example 2.Power >0.95 with AC<6 indicate strong predictive power of model.

FIG. 11 shows that release is driven by three main mechanisms:diffusion, dissolution, and depletion. Initial release (burst) will belargely driven by hydration of the gel and diffusion of protein from theouter portion of the matrix; diffusion will be limited by crosslinkingthat prevents hydration of encapsulated protein within the matrix. Fordissolution, once crosslinks are hydrolyzed by aqueous media, additionalprotein will become exposed, hydrated, and released from the matrixuntil the drug load is depleted. Release kinetics can be tuned to thetarget duration and desired shape by adjusting combinations of PEG MWand molar ratio.

FIG. 12 shows that Actual by Predicted scatter plot of the measured timeto 99% release vs. the predicted time to 99% release based on modelparameters. The goodness-of-fit for the predictive model for 99% releaseis indicated by RSquare Adj (>96%).

FIG. 13 shows that all three main factors have a statisticallysignificant impact (p<0.05) on time to total release (99%). Magnitude ofeach effect in rank order is: 1. Molar Ratio (continuous factor-negativecorrelation); 2. PEG-NHS molecular weight (categorical factor, i.e.,non-continuous or discrete); and 3. PEG-NH molecular weight (categoricalfactor). 40 kDa PEG-NH (highlighted in gray circles) has the largesteffect of PEG-NH reagents tested. 15 kDa PEG-NHS (highlighted in opencircles) has the largest effect of PEG-NHS reagents tested.

FIG. 14 is a graph showing that, in accordance with the predictivemodel, a combination of a molar ratio of 1.6, PEG-NH molecular weight(MW) of 10 kDa, and PEG-NHS MW of 10 kDa can produce a hydrogel with arelease period of 42.5 days for 99% release.

FIG. 15 is a graph showing that, in accordance with the predictivemodel, a combination of a molar ratio of 1.71, PEG-NH MW of 40 kDa, andPEG-NHS MW of 15 kDa can produce a hydrogel with a release period of 14days for 99% release.

FIG. 16 is a graph showing that, in accordance with the predictivemodel, a combination of a molar ratio of 1.54, PEG-NH MW of 40 kDa, andPEG-NHS MW of 15 kDa can produce a hydrogel with a release period of 21days for 99% release.

FIG. 17 is a graph showing that, in accordance with the predictivemodel, a combination of a molar ratio of 1.68, PEG-NH MW of 10 kDa, andPEG-NHS MW of 15 kDa can produce a hydrogel with a release period of 21days for 99% release. FIGS. 16-17 show that the same release period canbe targeted by selecting different combinations of PEG reagents andmolar ratios.

FIG. 18 is a graph showing that, in accordance with the predictivemodel, a combination of a molar ratio of 1.84, PEG-NH MW of 40 kDa, andPEG-NHS MW of 15 kDa can produce a hydrogel with a release period of8.88 days for 99% release.

FIG. 19 is a graph showing that, in accordance with the predictivemodel, a combination of a molar ratio of 1.30, PEG-NH MW of 15 kDa, andPEG-NHS MW of 10 kDa can produce a hydrogel with a release period of58.9 days for 99% release. FIGS. 18-19 show that by adjusting both themolar ratio and PEG reagents, release periods from about 8 to 59 dayscan be achieved.

FIG. 20 is a graph showing that, in accordance with the predictivemodel, a combination of a molar ratio of 1.30, PEG-NH MW of 40 kDa, andPEG-NHS MW of 15 kDa can produce a hydrogel with a release period of30.9 days for 99% release. FIGS. 18 and 20 show that by adjusting themolar ratio only, release periods within an allowable range for a givenPEG combination can be targeted.

FIG. 21 is a graph showing that, in accordance with the predictivemodel, a combination of a molar ratio of 1.83, PEG-NH MW of 20 kDa, andPEG-NHS MW of 20 kDa can produce a hydrogel with a release period of 30days for 99% release.

FIG. 22 is a graph showing that, in accordance with the predictivemodel, a combination of a molar ratio of 1.46, PEG-NH MW of 10 kDa, andPEG-NHS MW of 15 kDa can produce a hydrogel with a release period of 30days for 99% release. FIGS. 21 and 22 show that the same release periodcan be targeted by selecting different combinations of PEG reagents andmolar ratios.

DETAILED DESCRIPTION OF THE INVENTION

Cross-linked PEG hydrogels encapsulating micronized, solid-stateproteins have shown promise for sustained release on the order of weeksor months with good protein stability. Based on demonstrated loadingcapacities up to 88% w/w total solid loading, this approach is feasiblefor both low and medium dose molecules with durations on the order ofmonths, e.g., about 1 week to 6 months. In efforts to develop thissystem as a platform delivery system tunable for sustained releasesuitable for a variety of target product profiles, important formulationparameters impacting release kinetics have been identified and exploredin multifactorial studies. A method for non-linear modeling of releaseprofiles that captures the shape typical of degradable hydrogel releaseprofiles is introduced and utilized in evaluating the impact offormulation parameters on protein release.

The present invention is based in part on the discovery that thefollowing parameters are important in affecting the release profile of abiologic disposed in a hydrogel: (a) a molar ratio of a nucleophilicgroup to an electrophilic group, wherein a first precursor comprises twoor more nucleophilic groups and a second precursor comprises two or moreelectrophilic groups; (b) the number of the nucleophilic groups in thefirst precursor; (c) the number of the electrophilic groups in thesecond precursor; (d) the molecular weight of the first precursor; (e)the molecular weight of the second precursor; (f) a weight ratio of thebiologic and excipient to the hydrogel; (g) a weight percentage of thebiologic in a solid state formulation; and (h) a ratio of surface areato volume of the hydrogel. By adjusting at least one of these parameterswhile the other parameters are predetermined, one can produce an arrayof biologic-loaded hydrogel formulations with different releaseprofiles.

In some embodiments, a quantitative, predictive model can be used todetermine these parameters for a desired release profile. For example,using the predictive model, one can predetermine one or more of theabove-mentioned parameters, and adjust one or more parameter of interestto evaluate its effect on the release profile. The parameter of interestcan be adjusted until the desired release profile is achieved. Amongother things, the predictive model offers the ability to: (1) target thesame release period by selecting different combinations of precursorsand molar ratios; (2) target release periods, e.g., from 8-59 days, byadjusting both the molar ratio and precursors; and (3) target releaseperiods within an allowable range for a given precursor combination byadjusting the molar ratio only.

One aspect of the present disclosure relates to a method of producing ahydrogel with a desired release profile for a biologic disposed therein,wherein the biologic is in a solid state formulation prior to beingdisposed in the hydrogel, the method comprising: (i) predetermining atleast one of the following parameters: (a) the number of nucleophilicgroups in a first precursor; (b) the number of electrophilic groups in asecond precursor; (c) the molecular weight of the first precursor; (d)the molecular weight of the second precursor; (e) a weight ratio of thebiologic and excipient to the hydrogel; (f) a weight percentage of thebiologic in the solid state formulation; and (g) a ratio of surface areato volume of the hydrogel; (ii) determining a molar ratio of thenucleophilic group to the electrophilic group alone, or in combinationwith any one or more of the above parameters that is not predetermined,until the desired release profile is achieved; and (iii) crosslinkingthe first precursor and the second precursor at the determined molarratio around the solid state formulation under anhydrous conditions.

Another aspect of the present disclosure relates to a method ofproducing a hydrogel having a biologic disposed therein, wherein thebiologic is in a solid state formulation prior to being disposed in thehydrogel, and wherein the hydrogel is characterized by a desired releaseperiod of about one week to about six months for at least 90% biologicrelease, the method comprising: (i) selecting a first precursor thatcomprises two or more nucleophilic groups, wherein the first precursorhas a molecular weight in the range of about 1 kDa to about 100 kDa;(ii) selecting a second precursor that comprises two or moreelectrophilic groups, wherein the second precursor has a molecularweight in the range of about 1 kDa to about 100 kDa; (iii) determiningat least one of the following parameters alone, or in combination, untilthe desired release period is achieved: (a) a molar ratio of thenucleophilic group to the electrophilic group; (b) a weight ratio of thebiologic and excipient to the hydrogel; (c) a weight percentage of thebiologic in the solid state formulation; and (d) a ratio of surface areato volume of the hydrogel; and (iv) crosslinking the first precursor andthe second precursor at the determined molar ratio around the solidstate formulation under anhydrous conditions.

The precursors are not the hydrogels but can be crosslinked with eachother to form the hydrogel. Crosslinks can be formed by covalent bondsor physical bonds. Examples of physical bonds are ionic bonds,hydrophobic association of precursor molecule segments, andcrystallization of precursor molecule segments. The precursors can betriggered to react to form a crosslinked hydrogel. The precursors can bepolymerizable and include crosslinkers that are often, but not always,polymerizable precursors. Polymerizable precursors are thus precursorsthat have functional groups that can react with each other to formmatrices and/or polymers made of repeating units. Precursors may bepolymers.

To form covalently crosslinked hydrogels, the precursors are covalentlycrosslinked together. In general, polymeric precursors are polymers thatwill be joined to other polymeric precursors at two or more points, witheach point being a linkage to the same or different polymers. Precursorswith at least two reactive centers (for example, in free radicalpolymerization) can serve as crosslinkers since each reactive group canparticipate in the formation of a different growing polymer chain. Inthe case of functional groups without a reactive center, among others,crosslinking requires three or more such functional groups on at leastone of the precursor types. For instance, manyelectrophilic-nucleophilic reactions consume the electrophilic andnucleophilic functional groups so that a third functional group isneeded for the precursor to form a crosslink. Such precursors thus mayhave three or more functional groups and may be crosslinked byprecursors with two or more functional groups. A crosslinked moleculemay be crosslinked via an ionic or covalent bond, a physical force, orother attraction. A covalent crosslink, however, will typically offerstability and predictability in reactant product architecture.

In some embodiments, each precursor is multifunctional, meaning that itcomprises two or more electrophilic or nucleophilic functional groups,such that a nucleophilic functional group on one precursor may reactwith an electrophilic functional group on another precursor to form acovalent bond. At least one of the precursors comprises more than twofunctional groups, so that, as a result of electrophilic-nucleophilicreactions, the precursors combine to form crosslinked polymericproducts.

The precursors may be small molecules, such as acrylic acid or vinylcaprolactam, larger molecules containing polymerizable groups, such asacrylate-capped polyethylene glycol (PEG-diacrylate), or other polymerscontaining ethylenically-unsaturated groups, such as those of U.S. Pat.No. 4,938,763 to Dunn et al, U.S. Pat. Nos. 5,100,992 and 4,826,945 toCohn et al, or U.S. Pat. Nos. 4,741,872 and 5,160,745 to DeLuca et al.,the contents of each of which are hereby incorporated by referenceherein in their entireties to the extent they do not contradict what isexplicitly disclosed herein.

In some embodiments, each precursor comprises only nucleophilic or onlyelectrophilic functional groups, so long as both nucleophilic andelectrophilic precursors are used in the crosslinking reaction. Thus,for example, if a crosslinker has nucleophilic functional groups such asamines, the functional polymer may have electrophilic functional groupssuch as N-hydroxysuccinimides. On the other hand, if a crosslinker haselectrophilic functional groups such as sulfosuccinimides, then thefunctional polymer may have nucleophilic functional groups such asamines or thiols. Thus, functional polymers such as proteins, poly(allylamine), or amine-terminated di- or multifunctional poly(ethylene glycol)can be used.

The precursors may have biologically inert and hydrophilic portions,e.g., a core. In the case of a branched polymer, a core refers to acontiguous portion of a molecule joined to arms that extend from thecore, with the terminus of each arm having a functional group. Ahydrophilic precursor or precursor portion has a solubility of at least1 g/100 mL in an aqueous solution. A hydrophilic portion may be, forinstance, a polyether, for example, polyalkylene oxides such aspolyethylene glycol (PEG), polyethylene oxide (PEO), polyoxyethylene,polyethylene oxide-co-polypropylene oxide (PPO), co-polyethylene oxideblock or random copolymers, and polyvinyl alcohol (PVA), poly(vinylpyrrolidone) (PVP), poly(amino acids), dextran, or a protein. Theprecursors may have a polyalkylene glycol portion and may bepolyethylene glycol based, with at least about 80% or 90% by weight ofthe polymer comprising polyethylene oxide repeats. The polyethers andmore particularly poly(oxyalkylenes) or poly(ethylene glycol) orpolyethylene glycol are generally hydrophilic. As is customary in thesearts, the term PEG is used to refer to PEO with or without hydroxyl endgroups.

Precursors may be made with a hydrophobic portion provided that theresultant hydrogel retains the requisite amount of water. In some cases,the precursor is nonetheless soluble in water because it also has ahydrophilic portion. In other cases, the precursor makes dispersion inthe water (a suspension) but is nonetheless able to react to from acrosslinked material. Some hydrophobic portions may include a pluralityof alkyls, polypropylenes, or other groups. Some precursors withhydrophobic portions are sold under the trade names PLURONIC® F68,JEFFAMINE®, or TECTRONIC®. A hydrophobic molecule or a hydrophobicportion of a copolymer or the like is one that is sufficientlyhydrophobic to cause the molecule (e.g., polymer or copolymer) toaggregate to form micelles or microphases involving the hydrophobicdomains in an aqueous continuous phase or one that, when tested byitself, is sufficiently hydrophobic to precipitate from, or otherwisechange phase while within, an aqueous solution of water at pH from about7 to about 7.5 at temperatures from about 30 to about 50 degreescentigrade.

Precursors may have a plurality of arms, e.g., 2-100 arms, with each armhaving a terminus, bearing in mind that some precursors may bedendrimers or other highly branched materials. The terminus of each armcan include a nucleophilic or electrophilic group. An arm on a hydrogelprecursor refers to a linear chain of chemical groups that connect across-linkable functional group to a polymer core. Some embodiments areprecursors with between 3 and 300 arms; artisans will immediatelyappreciate that all the ranges and values within the explicitly statedranges are contemplated, e.g., 4 to 16, 8 to 100, or at least 6 arms.

In some embodiments, the first precursor can comprise about 2-30nucleophilic groups, e.g., about 2-25, about 2-20, about 2-15, about2-10, about 5-30, about 5-25, about 5-20, or about 5-15 nucleophilicgroups. In some embodiments, the first precursor comprises about 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30 nucleophilic groups.

In some embodiments, the second precursor can comprise about 2-30electrophilic groups, e.g., about 2-25, about 2-20, about 2-15, about2-10, about 5-30, about 5-25, about 5-20, or about 5-15 electrophilicgroups. In some embodiments, the second precursor comprises about 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30 electrophilic groups.

Thus hydrogels can be made, e.g., from a multi-armed precursor with afirst set of functional groups and another multi-armed precursor havinga second set of functional groups. For example, a six-armed oreight-armed precursor may have hydrophilic arms, e.g., polyethyleneglycol, terminated with primary amines, with the molecular weight of thearms being about 1,000 to about 40,000 Daltons (Da); artisans willimmediately appreciate that all ranges and values within the explicitlystated bounds are contemplated. Such precursors may be mixed withrelatively smaller precursors, for example, molecules with a molecularweight of between about 100 Da and about 5000 Da, or no more than about800 Da, 1000 Da, 2000 Da, or 5000 Da having at least about threefunctional groups, or between about 3 to about 16 functional groups;ordinary artisans will appreciate that all ranges and values betweenthese explicitly articulated values are contemplated. Such smallmolecules may be polymers or non-polymers and natural or synthetic.

In some embodiments, the precursors can be dendrimers. Dendrimers arehighly branched, radially symmetrical polymers in which the atoms arearranged in many arms and subarms radiating out from a central core. Insome embodiments, the precursors are not dendrimers.

Peptides may be used as precursors. In general, peptides with less thanabout 10 residues are preferred, although larger sequences (e.g.,proteins) may be used. Artisans will immediately appreciate that everyrange and value within these explicit bounds is included, e.g., 1-10,2-9, 3-10, 1, 2, 3, 4, 5, 6, or 7. Some amino acids have nucleophilicgroups (e.g., primary amines or thiols) or groups that can bederivatized as needed to incorporate nucleophilic groups orelectrophilic groups (e.g., carboxyls or hydroxyls).

Some hydrogels are made with a polyethylene glycol-containing precursor.Polyethylene glycol (PEG, also referred to as polyethylene oxide whenoccurring in a high molecular weight) refers to a polymer with a repeatgroup (CH₂CH₂O)_(n), with n being at least 3. A polymeric precursorhaving a polyethylene glycol thus has at least three of these repeatgroups connected to each other in a linear series. The polyethyleneglycol content of a polymer or arm is calculated by adding up all of thepolyethylene glycol groups on the polymer or arm, even if they areinterrupted by other groups. Thus, an arm having at least 1000 Da MWpolyethylene glycol has enough CH₂CH₂O groups to total at least 1000 DaMW. As is customary terminology in these arts, a polyethylene glycolpolymer does not necessarily refer to a molecule that terminates in ahydroxyl group. Molecular weights are abbreviated in thousands using thesymbol k, e.g., with 15 k Da meaning 15,000 Da molecular weight.Succinimidyl succinate, succinimidyl glutarate, succinimidyl adipate,and succinimidyl azelate are succinimidyl esters that have an estergroup that degrades by hydrolysis in water. Hydrolytically degradablethus refers to a material that would spontaneously degrade in vitro inan excess of water without any enzymes or cells present to mediate thedegradation. A time for degradation refers to effective disappearance ofthe material as judged by the naked eye. PEG and/or hydrogels, as wellas compositions that comprise the same, may be provided in a form thatis pharmaceutically acceptable, meaning that it is highly purified andfree of contaminants, e.g., pyrogens.

Non-limiting examples of nucleophilic groups include amine, hydroxyl,carboxyl, and thiol. In some embodiments, the nucleophilic group isamine. The amine can be a primary amine, a secondary amine, a tertiaryamine, or a cyclic amine. In some embodiments, the first precursorcomprises (aminopropyl)_(m) polyoxyethylene, wherein m is in the rangeof about 2 to about 10. For example, m is 2, 3, 4, 5, 6, 7, 8, 9, or 10.In some embodiments, the first precursor can be pentaerythritoltetra(aminopropyl) polyoxyethylene, hexaglycerol octa(aminopropyl)polyoxyethylene, or a combination thereof. The molecular weight of thefirst precursor can be in the range of about 1 kDa to about 100 kDa,e.g., about 5 kDa to about 100 kDa, about 10 kDa to about 100 kDa, orabout 20 kDa to about 100 kDa.

Non-limiting examples of electrophilic groups include sulfonyl chloride,chlorocarbonates, n-hydroxysucciniinidyl ester, succinimidyl ester,sulfasuccinimidyl esters, succinimide, succinimide ester,n-hydroxysuccinimide, maleimide, succinate, nitrophenyl carbonate,aldehyde, vinylsulfone, azide, hydrazide, isocyanate, diisocyanate,tosyl, tresyl, or carbonyldiimidazole, and those disclosed in U.S. Pat.Nos. 5,410,016 and 6,149,931, each of which are hereby incorporated byreference herein in its entirety to the extent they do not contradictwhat is explicitly disclosed herein. In some embodiments, theelectrophilic group is n-hydroxysucciniinidyl ester orn-hydroxysuccinimide. In some embodiments, the second precursorcomprises (succinimidyloxyglutaryl)_(n) polyoxyethylene, wherein n is inthe range of about 2 to about 10. For example, n is 2, 3, 4, 5, 6, 7, 8,9, or 10. In some embodiments, the second precursor can bepentaerythritol tetra(succinimidyloxyglutaryl) polyoxyethylene,pentaerythritol tetra(succinimidyloxysuccinyl) polyoxyethylene,pentaerythritol tetra(succinimidyl carboxypentyl) polyoxyethylene,hexaglycerol octa(succinimidyloxyglutaryl) polyoxyethylene, hexaglycerolocta(succinimidyloxysuccinyl) polyoxyethylene, or a combination thereof.The molecular weight of the second precursor can be in the range ofabout 1 kDa to about 100 kDa, e.g., about 5 kDa to about 100 kDa, about10 kDa to about 100 kDa, or about 20 kDa to about 100 kDa.

In some embodiments, the electrophilic group is n-hydroxysucciniinidylester or n-hydroxysuccinimide, and the nucleophilic group is primaryamine.

Certain functional groups, such as alcohols or carboxylic acids, do notnormally react with other functional groups, such as amines, underphysiological conditions (e.g., pH 7.2-11.0, 37° C.). However, suchfunctional groups can be made more reactive by using an activating groupsuch as N-hydroxysuccinimide. Certain activating groups includecarbonyldiimidazole, sulfonyl chloride, aryl halides, sulfasuccinimidylesters, N-hydroxysuccinimidyl ester, succinimidyl ester, epoxide,aldehyde, maleimides, imidoesters and the like.

The functional groups may be, e.g., electrophiles reactable withnucleophiles, groups reactable with specific nucleophiles, e.g., primaryamines, groups that form amide bonds with materials in the biologicalfluids, groups that form amide bonds with carboxyls, activated-acidfunctional groups, or a combination of the same. The functional groupsmay be, e.g., a strong electrophilic functional group, meaning anelectrophilic functional group that effectively forms a covalent bondwith a primary amine in aqueous solution at pH 9.0 at room temperatureand pressure and/or an electrophilic group that reacts by a Michael-typereaction. The strong electrophile may be of a type that does notparticipate in a Michael-type reaction or of a type that participates ina Michaels-type reaction. Examples of strong electrophiles that do notparticipate in a Michael-type reaction are: succinimides, succinimidylesters, or NHS-esters. Examples of Michael-type electrophiles areacrylates, methacrylates, methylmethacrylates, and other unsaturatedpolymerizable groups. More embodiments of the precursors and functionalgroups can be found at U.S. Pat. No. 9,205,150, the contents of whichare incorporated by reference in their entities.

The molar ratio of the nucleophilic group to the electrophilic group candetermine the crosslink density. A molar ratio of one results in thehighest crosslink density. A molar ratio of greater or less than one canlead to lower crosslink density than a molar ratio of one. The crosslinkdensity increases as the molar ratio increases until it reaches thevalue of one, then the crosslink density decreases as the molar ratioincreases beyond the value of one. The biologic embedded in the hydrogelcan be released faster when the crosslink density is lower. For example,see the results shown in FIG. 2. As a result, by adjusting the molarratio of the nucleophilic group to the electrophilic group, one can tunethe release kinetics of the biologic. Without wishing to be bound bytheory, the molar ratio effectively modulates both the crosslink density(i.e. number of covalent crosslinks forming the network) and the networkpore size within the hydrogel matrix. By decreasing the crosslinkdensity, the effective pore size of the matrix can be increased,resulting in faster diffusion of protein through the matrix.Additionally, decreasing crosslink density can increase domains in thehydrogel where the local concentration of PEG surrounding the proteinparticle is insufficient to retain protein in the solid state uponhydration, leading to an increase in burst release as well as the rateof diffusion on a mass basis. Thus it is expected that as molar ratioincreases, both burst release and release kinetics in the diffusioncontrolled regime will increase. Additionally, there is an observedincrease in the slope of the release profile in thedissolution-controlled regime with increasing molar ratio. This can beexplained by the reduction in the extent of cross-linking withincreasing molar ratio as there is a greater mismatch between the numberof nucleophilic groups and electrophilic groups available to react andcrosslink. The growth rate is determined by the rate of hydrolysis ofthe crosslinks, leading to an increase in hydrogel swelling and aconcomitant decrease in local concentrations of PEG that result inadditional dissolution of protein particles. Swelling of the hydrogel isalso correlated with hydrogel porosity, which is increased as proteindissolution occurs. As molar ratio increases and protein dissolutionupon hydration increases, so will the effective porosity of thehydrogel, leading to more swelling, a faster hydrolysis, and fastergrowth rate in the dissolution-controlled regime. Furthermore, theinflection point identifying the transition between diffusion anddissolution-controlled regimes will be inversely correlated with molarratio. As effective rates of diffusion increase with increasing molarratio, the time it takes for diffusion to increase to the extent that itis no longer the rate-limiting step decreases, and thereby shifts theinflection point to earlier time points. Considered comprehensively,varying the molar ratio alone can permit the release profile to be tunedfrom near linear to sigmoidal depending on the desired result.

In some embodiments, the molar ratio of the nucleophilic group to theelectrophilic group is greater than 1. In some embodiments, the molarratio of the nucleophilic group to the electrophilic group is lessthan 1. In some embodiments, the molar ratio of the nucleophilic groupto the electrophilic group can be in the range of about 0.1 to 3.0,e.g., about 0.1 to 0.9, about 0.1 to 0.8, about 0.1 to 0.7, about 0.1 to0.6, about 0.2 to 0.9, about 0.2 to 3.0, about 0.2 to 2.8, about 0.2 to2.5, about 0.5 to 2.5, about 0.5 to 2.0, about 0.8 to 2.5, about 0.8 to2.0, about 1.1 to 2.0, about 1.1 to 2.5, about 1.1 to 3.0, about 1.5 to3.0, about 1.5 to 2.5, about 1.5 to 2.0, or about 1.3 to 1.8. In someembodiments, the molar ratio of the nucleophilic group to theelectrophilic group can be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2,2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0. In some embodiments, themolar ratio of the nucleophilic group to the electrophilic group is not1.

In the predictive model, the molar ratio has a continuous effect onrelease profile. As used herein, the term “continuous” means that onecan interpolate between levels. As such, molar ratios can be adjustedcontinuously to give incremental variation in release periods. In someembodiments, the release period in the release profile can be adjustedat a rate of about −41 days per molar ratio change when the molar ratiois greater than 1, e.g., in the range of about 1.3 to about 1.8. In someembodiments, the release period in the release profile can be adjustedat a rate of about 103 days per molar ratio change when the molar ratiois less than 1, e.g., in the range of about 0.77 to about 0.56.

Related to the molar ratio, the number of the nucleophilic group in thefirst precursor and/or the number of the electrophilic group in thesecond precursor can also determine the crosslink density. Generally, ata given molar ratio, the higher the number of the nucleophilic orelectrophilic group, the higher the crosslink density. In someembodiments, the method comprises selecting 8-arm PEG NH and 8-arm PEGNHS reagents for a release period of 60 days or longer. In someembodiments, the method comprises selecting 4-arm PEG NH and 4-arm PEGNHS reagents for a release period of less than 60 days.

Another parameter that one can use to tune the release kinetics is themolecular weight of the first and/or second precursor. At a given molarratio, the lower the molecular weight of the precursor, the smaller thenetwork pore size. The molecular weights of the first and secondprecursors have a non-continuous or discrete effect on release profilein the predictive model described herein. As used herein, the term“non-continuous” or “discrete” means that one cannot interpolate betweenlevels. For example, a combination of first and second precursors (e.g.,PEG reagents) with predetermined molecular weights can define a range ofrelease periods possible. Other factors such as molar ratio can be usedto fine tune the release profile or release period. For example, asshown in Table 9, a combination of 10 kDa PEG NH and 15 kDa PEG NHSdefines a release period in the range of 9-31 days; by varying the molarratio in the range of 1.3 and 1.8, one can continuously tune the releaseperiod from 31 days to 9 days. In the predictive model, the molar ratio,molecular weight of the first precursor, and molecular weight of thesecond precursor are independent parameters.

In some embodiments, the method comprises: (a) selecting a range ofmolar ratios defined by a first molar ratio as minimum and a secondmolar ratio as maximum, and the molecular weights of the first and thesecond precursors, thereby resulting in a range of release periodsdefined by a first release period as maximum and a second release periodas minimum, wherein the first molar ratio is 1 or greater, and whereinthe desired release period is within the range of release periods; and(b) determining the desired molar ratio in accordance with either one ofthe following formulae:

Desired molar ratio=First molar ratio+(First release period −Desiredrelease period)/41; and

Desired molar ratio=Second molar ratio+(Second release period −Desiredrelease period)/41.

In some embodiments, the range of molar ratios is about 1.3 to about 1.8or a subrange thereof. In some embodiments, the first molar ratio can beabout 1.3, and the second molar ratio can be greater than about 1.3 andno more than about 1.8, e.g., about 1.4, about 1.5, about 1.6, about1.7, or about 1.8. In some embodiments, the first molar ratio can beabout 1.4, and the second molar ratio can be greater than about 1.4 andno more than about 1.8, e.g., about 1.5, about 1.6, about 1.7, or about1.8. In some embodiments, the first molar ratio can be about 1.5, andthe second molar ratio can be greater than about 1.5 and no more thanabout 1.8, e.g., about 1.6, about 1.7, or about 1.8. In someembodiments, the first molar ratio can be about 1.6, and the secondmolar ratio can be greater than about 1.6 and no more than about 1.8,e.g., about 1.7 or about 1.8. In some embodiments, the first molar ratiocan be about 1.7, and the second molar ratio can be greater than about1.7 and no more than about 1.8, e.g., about 1.8.

In some embodiments, the method comprises: (a) selecting a range ofmolar ratios defined by a first molar ratio as maximum and a secondmolar ratio as minimum, and the molecular weights of the first and thesecond precursors, thereby resulting in a range of release periodsdefined by a first release period as maximum and a second release periodas minimum, wherein the first molar ratio is 1 or less, and wherein thedesired release period is within the range of release periods; and (b)determining the desired molar ratio in accordance with either one of thefollowing formulae:

Desired molar ratio=First molar ratio −(First release period −Desiredrelease period)/103; and

Desired molar ratio=Second molar ratio −(Second release period −Desiredrelease period)/103.

In some embodiments, the range of molar ratios is about 0.77 to about0.56 or a subrange thereof. In some embodiments, the first molar ratiocan be about 0.77, and the second molar ratio can be less than about0.77 and no less than about 0.56, e.g., about 0.7, about 0.65, about0.6, or about 0.56. In some embodiments, the first molar ratio can beabout 0.7, and the second molar ratio can be less than about 0.7 and noless than about 0.56, e.g., about 0.65, about 0.6, or about 0.56. Insome embodiments, the first molar ratio can be about 0.65, and thesecond molar ratio can be less than about 0.65 and no less than about0.56, e.g., about 0.6 or about 0.56. In some embodiments, the firstmolar ratio can be about 0.6, and the second molar ratio can be lessthan about 0.6 and no less than about 0.56, e.g., about 0.56.

Another parameter that one can use to tune the release kinetics is aweight ratio of the biologic and excipient to the hydrogel. The weightratio of the biologic and excipients to the hydrogel is also referred toherein as “solid loading.” This refers to the weight ratio of thebiologic and excipient to the total weight of the biologic, excipient,and polymer comprising the protein-loaded hydrogel. It was discoveredthat increasing the solid loading can change the shape of the releaseprofile, primarily due to faster release during the initial diffusionphase before the inflection point. There is also likely an inversecorrelation between the onset of the dissolution phase (i.e. theinflection point) and the solid loading. Without wishing to be bound bytheory, with increased solid loading, faster release during the initialdiffusion phase is expected because there will be a larger quantity ofprotein in microenvironments of relatively low PEG concentration whereprotein solubility is less limited. Protein in such regions will bedissolved upon initial hydration of the matrix, increasing the rate ofconcentration-dependent diffusion. Additionally, the increase in proteinparticles dissolved upon initial hydration will create voids within thematrix and lead to an increase in matrix porosity. A more porous matrixwill also increase the effective rate of diffusion through the bulkmatrix and released as it reaches the surface. Without wishing to bebound by theory, the inverse correlation between inflection point andsolid loading can also be hypothetically explained by the increase inrate of diffusion observed with increased solid loading. The inflectionpoint signifies the transition between a diffusion-controlled regime anda dissolution-controlled regime. As solid loading increases, thediffusion rate starts higher and increases more rapidly, leading to ashorter duration before diffusion is no longer rate limiting. In thediffusion-controlled regime, diffusion of dissolved protein through thematrix is the rate-limiting step for protein release. As more proteinparticles dissolve, voids within the matrix are created and the porosityof the matrix increases, leading to increased rates of diffusion. In thedissolution-controlled regime, diffusion through the matrix is no longerthe rate-limiting step. This point is reached more rapidly as solidloading increases. For example, FIG. 5 illustrates the effects of solidloading on the release rate and profile.

In some embodiments, the biologic can be a peptide or protein, such as arecombinant protein. The recombinant protein can be an antibody orfragment thereof, a short chain variable fragment (scFv), a growthfactor, an angiogenic factor, or insulin. Other water soluble biologicsare carbohydrates, polysaccharides, nucleic acids, antisense nucleicacids, RNA, DNA, small interfering RNA (siRNA), and aptamers. In someembodiments, the biologic is an anti-vascular endothelial growth factoragent such as aflibercept, bevacizumab, and ranibizumab. In someembodiments, the biologic is an immunoglobulin G such as IgG1, IgG2,IgG3, and IgG4. In some embodiments, the biologic is a bispecificmonoclonal antibody. There are many formats of bispecific monoclonalantibody, but the two main categories are IgG-like and non-IgG-like. Insome embodiments, the biologic is a fusion protein with decoy receptordomains.

The water-soluble biologics can be prepared as particles beforedispersal into the hydrogels. Multiple protein particulationtechnologies, such as spray drying or precipitation exist and may beemployed provided the protein of interest is compatible with suchprocessing. An embodiment of particle preparation involves receiving thebiologic without substantial denaturation, e.g., from a supplier oranimal or recombinant source. The solid phase is a stable form for theprotein. The protein is lyophilized or concentrated or used as received.The protein is then prepared as a fine powder without denaturation byprocessing it in a solid state and avoiding high temperatures, moisture,and optionally in an oxygen free environment. Powders may be preparedby, for example, spray drying, grinding, ball milling, cryomilling,microfluidizing or mortar-and-pestle followed by sieving a solidprotein. The protein may also be processed in a compatible anhydrousorganic solvent in which the protein in question is not soluble, whilekeeping the protein in a solid form. Particle size reduction to thedesired range may be achieved by, for example, grinding, ball milling,jet milling of a solid protein suspension in a compatible organicsolvent.

Common excipients include, but are not limited to, sucrose, proline,trehalose, trileucine, mannitol, isoleucine, buffers such as histidine,phosphate, acetate, and polysorbates.

The solid loading can be in the range of 0.1 to 0.9, e.g., 0.1-0.8,0.1-0.7, 0.1-0.6, 0.2-0.9, 0.2-0.8, 0.2-0.7, 0.2-0.6, 0.3-0.9, 0.3-0.8,0.3-0.7, or 0.3-0.6. In some embodiments, the solid loading can be about0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9.

Yet another parameter that one can use to tune the release kinetics isthe weight percentage of the biologic in a solid state formulation. Asolid state formulation described herein is compositionally differentfrom a dehydrated hydrogel of the present disclosure. The dehydratedhydrogel is formed by crosslinking a first precursor and a secondprecursor around the solid state formulation. The weight percentage ofthe biologic in a solid state formulation is also referred to herein as“protein content.” It was discovered that the higher the proteincontent, the faster the rate of release. In addition, the proteincontent can also impact the release profile shape by changing theinflection point and release rate up to the inflection point. Theprotein content of the micronized particle can determine the effectiveconcentration of protein in the microenvironment upon dissolution of theparticle within the matrix. Diffusion within the PEG hydrogel is drivenby such concentration gradients within the microenvironments throughoutthe matrix. Thus, for solid state formulations with high proteincontent, the local concentrations upon dissolution will be higher thanfor formulations with low protein content, which creates a greaterdriving force for diffusion. This is observed by the correlation betweenthe rate of release in the diffusion-controlled regime prior to theinflection point and protein content. Without wishing to be bound bytheory, the inverse correlation between the inflection point and theprotein content can be explained by reducing the time frame in which theconcentration-dependent diffusion rate increases and dissolution becomesthe rate-limiting step for release. For example, FIG. 4 illustrates theeffects of protein content on the release rate and profile.

The solid state formulation comprises the biologic and one or moreexcipients. The solid state formulation can be produced by methods suchas precipitation, crystallization, lyophilizing, spray drying, milling,microtemplating, spray freezing, reversible precipitation, supercritical fluid drying, and electrospraying. Lyophilized, spray dried orotherwise processed proteins are often formulated with sugars such astrehalose or sucrose to stabilize the protein or other processes used toprepare the proteins. These sugars may be allowed to persist in theparticle throughout the hydrogel formation process. The protein contentcan be in the range of 10% to 95% by weight, e.g., 10%-90%, 10%-80%,10%-70%, 10%-60%, 20%-90%, 20%-80%, 20%-70%, 30%-90%, 30%-80%, or30%-70% by weight. In some embodiments, the protein content can be about10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% by weight.

The solid state formulation can include particles having an averagediameter no more than 20 μm. In some embodiments, the solid stateformulation can include particles having an average diameter of about 10nm to 20 μm. For example, the particles can have an average diameter ofabout 10 nm to 15 μm, 10 nm to 10 μm, 50 nm to 20 μm, 50 nm to 15 μm, 50nm to 10 μm, 100 nm to 15 μm, 100 nm to 10 μm, 200 nm to 10 μm, 400 nmto 10 μm, 600 nm to 10 μm, 1 μm to 10 μm, 2 μm to 10 μm, 100 nm to 1 μm,or 200 nm to 800 nm.

Yet another parameter that one can use to tune the release kinetics isthe ratio of surface area to volume of the hydrogel. It was discoveredthat the higher the ratio of surface area to volume, the faster the rateof release. For example, FIG. 6 illustrates the differences in releaserate for two different forms (slab and microparticles). The ratio can bechanged by altering the form factor of the hydrogel. Exemplary formfactors include, but are not limited to, slabs, microflakes,microparticles, and powder. Methods of measuring the surface area of amaterial is known in the art, including the Brunauer Emmett Teller (BET)model. The ratio of surface area to volume can be in the range of about1-150 mm⁻¹, e.g., about 2-100 mm⁻¹, about 5-100 mm⁻¹, or about 10-75mm⁻¹.

When one of the eight parameters is predetermined, one can adjust atleast one of the remaining seven parameters to achieve a desired releaseprofile. For example, when the weight ratio of the biologic andexcipient to the hydrogel is predetermined, one can adjust at least oneof the following parameters: (a) the molar ratio of the nucleophilicgroup and the electrophilic group; (b) the number of the nucleophilicgroups in the first precursor; (c) the number of the electrophilicgroups in the second precursor; (d) the molecular weight of the firstprecursor; (e) the molecular weight of the second precursor; (f) aweight percentage of the biologic in a solid state formulation; and (g)a ratio of surface area to volume of the hydrogel.

In some embodiments, when two of the eight parameters are predetermined,one can adjust at least one of the remaining six parameters to achieve adesired release profile.

In some embodiments, when three of the eight parameters arepredetermined, one can adjust the remaining five parameters to achieve adesired release profile.

In some embodiments, when four of the eight parameters arepredetermined, one can adjust at least one of the remaining fourparameters to achieve a desired release profile.

In some embodiments, when five of the eight parameters arepredetermined, one can adjust at least one of the remaining threeparameters to achieve a desired release profile.

In some embodiments, when six of the eight parameters are predetermined,one can adjust at least one of the remaining two parameters to achieve adesired release profile.

In some embodiments, when seven of the eight parameters arepredetermined, one can adjust the remaining parameter to achieve adesired release profile.

A predictive model can be used for determining one or more of theparameters. In some embodiments, after one determines the desiredrelease period and the molecular weights of the first and secondprecursors, the predictive model can provide the molar ratio that willresult in the desired release period.

The desired release profile can depend on a variety of factorsincluding, but not limited to, the biologic, the disease or conditionbeing treated, and the administration route. In some embodiments, thedesired release profile comprises a release period of about one week tosix months for at least 90% biologic release, e.g., about two months tosix months or about one week to two months. For ocular applications, thedesired release profile can include controlled release for about 14days. In some embodiments, the desired release profile exhibitsnear-linear release for about 1 week to 6 months. In some embodiments,the desired release profile exhibits near-linear release over at leastone week. In some embodiments, the desired release profile exhibitsnear-linear release over at least two weeks. In some embodiments, thedesired release profile exhibits near-linear release over at least onemonth. In some embodiments, the desired release profile exhibitsnear-linear release over at least two months. In some embodiments, thedesired release profile exhibits near-linear release over at least sixmonths.

A desired release profile can include a delayed-release portion, asigmoidal shape, a linear portion, a non-linear portion, a logarithmicportion, an exponential portion, or a combination thereof. In someembodiments, a desired release profile may be a sigmoidal releaseprofile with an extended delay with minimal or no release followed by asustained release until depletion. Such a release profile might bedesirable in combination with a liquid loading dose and tuned to beginthe sustained release upon clearance of the initial loading dose tobelow effective levels from the body. Another desired release profilemay be achieved through simultaneous administration of two or morehydrogels with tuned sigmoidal profiles to achieve a pulsatile releaseprofile. Yet another desired release profile may be achieved throughsimultaneous administration of two or more hydrogels with differentrelease profiles where one is fast (e.g., near linear release,logarithmic, or exponential) and another slower/delayed (sigmoidal).

Hydrogel Formation

In the presence of an anhydrous and hydrophobic solvent, the hydrogelcan be formed by crosslinking the first precursor and the secondprecursor at the determined molar ratio around the solid stateformulation having the biologic. In some embodiments, the anhydrous andhydrophobic solvent can be methylene chloride, ethyl acetate, dimethylcarbonate, chloroform, or a combination thereof.

Some precursors react using initiators. An initiator group is a chemicalgroup capable of initiating a free radical polymerization reaction. Forinstance, it may be present as a separate component, or as a pendentgroup on a precursor. Initiator groups include thermal initiators,photoactivatable initiators, and oxidation-reduction (redox) systems.Long wave UV and visible light photoactivatable initiators include, forexample, ethyl eosin groups, 2,2-dimethoxy-2-phenyl acetophenone groups,other acetophenone derivatives, thioxanthone groups, benzophenonegroups, and camphorquinone groups. Examples of thermally reactiveinitiators include 4,4′ azobis(4-cyanopentanoic acid) groups, andanalogs of benzoyl peroxide groups. Several commercially available lowtemperature free radical initiators, such as V-044, available from WakoChemicals USA, Inc., Richmond, Va., may be used to initiate free radicalcrosslinking reactions at body temperatures to form hydrogel coatingswith the aforementioned monomers.

Metal ions may be used either as an oxidizer or a reductant in redoxinitiating systems. For example, ferrous ions may be used in combinationwith a peroxide or hydroperoxide to initiate polymerization, or as partsof a polymerization system. In this case, the ferrous ions would serveas a reductant. Alternatively, metal ions may serve as an oxidant. Forexample, the ceric ion (4+ valence state of cerium) interacts withvarious organic groups, including carboxylic acids and urethanes, toremove an electron to the metal ion, and leave an initiating radicalbehind on the organic group. In such a system, the metal ion acts as anoxidizer. Potentially suitable metal ions for either role are any of thetransition metal ions, lanthanides and actinides, which have at leasttwo readily accessible oxidation states. Particularly useful metal ionshave at least two states separated by only one difference in charge. Ofthese, the most commonly used are ferric/ferrous; cupric/cuprous;ceric/cerous; cobaltic/cobaltous; vanadate V vs. IV; permanganate; andmanganic/manganous. Peroxygen-containing compounds, such as peroxidesand hydroperoxides, including hydrogen peroxide, t-butyl hydroperoxide,t-butyl peroxide, benzoyl peroxide, cumyl peroxide may be used.

An example of an initiating system is the combination of a peroxygencompound in one solution, and a reactive ion, such as a transitionmetal, in another. In this case, no external initiators ofpolymerization are needed and polymerization proceeds spontaneously andwithout application of external energy or use of an external energysource when two complementary reactive functional groups containingmoieties interact at the application site.

A visualization agent may be used as a powder in a hydrogel; it reflectsor emits light at a wavelength detectable to a human eye so that a userapplying the hydrogel could observe the object when it contains aneffective amount of the agent. Agents that require a machine aid forimaging are referred to as imaging agents herein, and examples includeradioopaque contrast agents and ultrasound contrast agents.

Some biocompatible visualization agents are FD&C BLUE #1, FD&C BLUE #2,and methylene blue. These agents are preferably present in the finalelectrophilic-nucleophilic reactive precursor species mix at aconcentration of more than 0.05 mg/ml and preferably in a concentrationrange of at least 0.1 to about 12 mg/ml, and more preferably in therange of 0.1 to 4.0 mg/ml, although greater concentrations maypotentially be used, up to the limit of solubility of the visualizationagent. Visualization agents may be covalently linked to the molecularnetwork of the hydrogel, thus preserving visualization after applicationto a patient until the hydrogel hydrolyzes to dissolution.

Visualization agents may be selected from among any of the variousnon-toxic colored substances suitable for use in medical implantablemedical devices, such as FD&C BLUE dyes 3 and 6, eosin, methylene blue,indocyanine green, or colored dyes normally found in synthetic surgicalsutures. Reactive visualization agents such as NHS-fluorescein can beused to incorporate the visualization agent into the molecular networkof the hydrogel. The visualization agent may be present with eitherreactive precursor species, e.g., a crosslinker or functional polymersolution. The preferred colored substance may or may not becomechemically bound to the hydrogel. The visualization agent may be used insmall quantities, e.g., 1% weight/volume, more preferably less than0.01% weight/volume and most preferably less than 0.001% weight/volumeconcentration; artisans will immediately appreciate that all the rangesand values within the explicitly stated ranges are contemplated. Theagent tends to mark the location of the particle and provides anindication of its presence and dissolution rate.

The dehydrated hydrogel may be formed from the cross-linked hydrogel sothat, upon hydration in physiological solution, a hydrogel is formedthat is water-degradable, as measurable by the hydrogel losing itsmechanical strength and eventually dissipating in vitro in an excess ofwater by hydrolytic degradation of water-degradable groups. This test ispredictive of hydrolytically-driven dissolution in vivo, a process thatis in contrast to cell or protease-driven degradation. Significantly,however, polyanhydrides or other conventionally-used degradablematerials that degrade to acidic components tend to cause inflammationin tissues. The hydrogels, however, may exclude such materials, and maybe free of polyanhydrides, anhydride bonds, or precursors that degradeinto acid or diacids.

For example, electrophilic groups such as N-hydroxysuccinimidylglutarate, N-hydroxysuccinimidyl succinate, N-hydroxysuccinimidylcarbonate, N-hydroxysuccinimidyl adipate or N-hydroxysuccinimidylazelate may be used and have esteric linkages that are hydrolyticallylabile. More linear hydrophobic linkages such as pimelate, suberate,azelate or sebacate linkages may also be used, with these linkages beingless degradable than succinate, glutarate or adipate linkages. Branched,cyclic or other hydrophobic linkages may also be used. Polyethyleneglycols and other precursors may be prepared with these groups. Thecrosslinked hydrogel degradation may proceed by the water-drivenhydrolysis of the biodegradable segment when water-degradable materialsare used. Polymers that include ester linkages may also be included toprovide a desired degradation rate, with groups being added orsubtracted near the esters to increase or decrease the rate ofdegradation. Thus it is possible to construct a hydrogel with a desireddegradation profile, from a few days to many months, using a degradablesegment. If polyglycolate is used as the biodegradable segment, forinstance, a crosslinked polymer could be made to degrade in about 1 toabout 30 days depending on the crosslinking density of the network.Similarly, a polycaprolactone based crosslinked network can be made todegrade in about 1 to about 8 months. The degradation time generallyvaries according to the type of degradable segment used, in thefollowing order: polyglycolate<polylactate<polytrimethylenecarbonate<polycaprolactone. Thus it is possible to construct a hydrogelwith a desired degradation profile, from a few days to many months,using a degradable segment.

A biodegradable linkage in the hydrogel and/or precursor may bewater-degradable or enzymatically degradable. Illustrativewater-degradable biodegradable linkages include polymers, copolymers andoligomers of glycolide, dl-lactide, 1-lactide, dioxanone, esters,carbonates, and trimethylene carbonate. Illustrative enzymaticallybiodegradable linkages include peptidic linkages cleavable bymetalloproteinases and collagenases. Examples of biodegradable linkagesinclude polymers and copolymers of poly(hydroxy acid)s,poly(orthocarbonate)s, poly(anhydride)s, poly(lactone)s,poly(aminoacid)s, poly(carbonate)s, and poly(phosphonate)s.

If it is desired that a biocompatible crosslinked matrix bebiodegradable or absorbable, one or more precursors having biodegradablelinkages present in between the functional groups may be used. Thebiodegradable linkage optionally also may serve as the water solublecore of one or more of the precursors used to make the matrix. For eachapproach, biodegradable linkages may be chosen such that the resultingbiodegradable biocompatible crosslinked polymer will degrade or beabsorbed in a desired period of time.

Matrix materials may be chosen so that degradation products are absorbedinto the circulatory system and essentially cleared from the body viarenal filtration. The matrix materials may be hydrogels in aphysiological solution. One method is to choose precursors that are notbroken down in the body, with linkages between the precursors beingdegraded to return the precursors or precursors with small changescaused by the covalent crosslinking process. This approach is incontrast to choosing biological matrix materials that are destroyed byenzymatic processes and/or materials cleared by macrophages, or thatresult in by-products that are effectively not water soluble. Materialsthat are cleared from the body by renal filtration can be labeled anddetected in the urine using techniques known to artisans. While theremight be at least a theoretical loss of some of these materials to otherbodily systems, the normal fate of the material is a kidney clearanceprocess. The term “essentially cleared” thus refers to materials thatare normally cleared through the kidneys.

Applications

In some embodiments, a hydrogel material may be placed into the patient,e.g., in a tissue or organ, including intraocularly, intravitreally,superchoroidally, subconjunctivally, topically, subcutaneously,intramuscularly, intraperitoneally, in a potential space of a body, orin a natural cavity or opening. The material provides a depot forrelease of a therapeutic agent (e.g., a biologic) over time. Embodimentsthus include between about 0.05 and about 500 ml volumes for placement(referring to total volume in the case of particle collectionsdelivered); artisans will immediately appreciate that all the ranges andvalues within the explicitly stated ranges are contemplated, e.g., from1 to 10 ml or from 5 to 50 ml. Intraperitoneal or intramuscularinjection, for instance, is a useful area for extended control releaseof agents over hours, days, or weeks.

The materials described herein may be used to deliver drugs or othertherapeutic agents (e.g., imaging agents or markers). One mode ofapplication is to apply a mixture of hydrogel particles and othermaterials (e.g., therapeutic agent, buffer, accelerator, initiator)through a needle, cannula, catheter, or hollow wire to a site. Themixture may be delivered, for instance, using a manually controlledsyringe or mechanically controlled syringe, e.g., a syringe pump.Alternatively, a dual syringe or multiple-barreled syringe ormulti-lumen system may be used to mix the hydrogel particles at or nearthe site with a hydrating fluid and/or other agents.

The dehydrated hydrogels may be provided in flowable form to the site,e.g., as flowable particles. The hydrogels may be suspended in a liquidand applied to the site. The hydrogel particles may be made to have amaximum diameter for manual passage out of a syringe through a 3 to 5French catheter, or a 10 to 30 gauge needle. Artisans will immediatelyappreciate that all the ranges and values within the explicitly statedranges are contemplated, e.g., 25 to 30 gauge. The use of small needlesis particularly advantageous in the eye, which is a sensitive organ.Applications to other organs are also advantageous, e.g., to controlbleeding or other damage. The particles may be formed by creating ahydrogel and then breaking it up into smaller pieces. The hydrogel maybe, e.g., ground in a ball mill or with a mortar and pestle, or choppedor diced with knives or wires. Or the hydrogel may be cut up in ablender or similar apparatus. The hydrogel may also be forced through amesh or casted into a template mold with desired size and shape. Thehydrogel may contain the therapeutic agent-loaded particles. Some or allof the hydrogel particles may contain the therapeutic agent-loadedparticles. In some embodiments, a first set of therapeutic agent-loadedparticles loaded with a first therapeutic agent is included inside afirst set of hydrogel particles and a second set of therapeuticagent-loaded particles loaded with a second therapeutic agent isincluded inside a second set of hydrogel particles. In this manner, aplurality of therapeutic agents may be released from a single implant.Embodiments of the particles include those with a particular shape suchas sphere, rod, or disc.

Embodiments include placement of a plurality of hydrogel particles. Thehydrogel particles may comprise a therapeutic agent. The particles maybe made with a size for manual passage through a 25-gauge or smallerdiameter needle. The pressure to force the particles through the needlemay be provided manually.

An alternative to delivery of particles is to pre-form the gel as ashaped article and then introduce the material into the body. Forexample, the hydrogels may be formed as spheres, rods, cylinders, orother shapes. Embodiments include solid rods of hydrogels forsubcutaneous implantation and delivery of one or more therapeuticagents.

Hydrogels as set forth herein may be used for tissue augmentation. Theuse of collagen for dermal augmentation is well known. Hydrogels, forexample, may be used for dermal filler or for tissue augmentation.Embodiments include injecting or otherwise placing a plurality ofparticles in a tissue, or forming a hydrogel in situ. The material maybe injected or otherwise placed at the intended site.

Hydrogels as set forth herein may be used to separate tissues to reducea dose of radioactivity received by one of the tissues. As set forth inU.S. Pat. No. 7,744,913, which is hereby incorporated by referenceherein for all purposes with the present specification controlling incase of conflict, spacer materials may be placed in a patient. Certainembodiments are a method comprising introducing a spacer to a positionbetween a first tissue location and a second tissue location to increasea distance between the first tissue location and the second tissuelocation. Further, there may be a step of administering a dose ofradioactivity to at least the first tissue location or the second tissuelocation. A method, for example, is delivering a therapeutic dose ofradiation to a patient comprising introducing a biocompatible,biodegradable particulate hydrogel, e.g., a collection of particlesoptionally with radioopaque contents, between a first tissue locationand a second tissue location to increase a distance between the firsttissue location and the second tissue location, and treating the secondtissue location with the therapeutic dose of radiation so that thepresence of the filler device causes the first tissue location toreceive less of the dose of radioactivity compared to the amount of thedose of radioactivity the first tissue location would receive in theabsence of the spacer. The spacer may be introduced as a dehydratedhydrogel that forms a hydrogel in the patient that is removed bybiodegradation of the spacer-hydrogel in the patient. An example is thecase wherein the first tissue location is associated with the rectum andthe second tissue location is associated with the prostate gland. Theamount of reduction in radiation can vary. Embodiments include at leastabout 10% to about 90%; artisans will immediately appreciate that allthe ranges and values within the explicitly stated ranges arecontemplated, e.g., at least about 50%. The radiation may alternativelybe directed to a third tissue so that the first tissue or the secondtissue received a lower amount of radiation as a result of itsseparation from the other tissue(s). The first tissue and the secondtissue may be adjacent to each other in the body, or may be separatedfrom each other by other tissues. Spacer volumes for separating tissuesare dependent on the configuration of the tissues to be treated and thetissues to be separated from each other. In many cases, a volume ofabout 20 cubic centimeters (cc's or mls) is suitable. In otherembodiments, as little as about 1 cc might be needed. Other volumes arein the range of about 5-1000 cc; artisans will immediately appreciatethat all the ranges and values within the explicitly stated ranges arecontemplated, e.g., 10-30 cc. In some embodiments, spacers areadministered in two doses at different times so as to allow the tissuesto stretch and accommodate the spacer and thereby receive a largervolume of spacer than would otherwise be readily possible. Tissues to beseparated by a spacer include, for example, at least one of a rectum,prostate, and breast, or a portion thereof. For instance, a firstportion of a breast may be separated from a second portion.

The details of the invention are set forth in the accompanyingdescription below. Although methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent invention, illustrative methods and materials are now described.Other features, objects, and advantages of the invention will beapparent from the description and from the claims. In the specificationand the appended claims, the singular forms also include the pluralunless the context clearly dictates otherwise. Unless defined otherwise,all technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. All patents and publications cited in thisspecification are incorporated herein by reference in their entireties.

Definitions

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) mayrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

As used herein, the terms “release profile” and “release kinetics” areused interchangeably to refer to the manner a biologic is released fromthe hydrogel under physiological conditions as a function of time. Arelease profile can be characterized by a release period and one or morerelease rates during the release period. A release profile can bevisualized by a graph having time on the X-axis and a measure ofbiologic release on the Y-axis (e.g. percentage, cumulative mass ofreleased biologic, or ratio of cumulative mass of released biologic tototal hydrogel mass).

As used herein, the term “near-linear release” refers to a release rateproportional to t^(n), where t is time, and n is in the range of 0.5-1.Artisans will immediately appreciate that all the ranges and valueswithin the explicitly stated ranges are contemplated, e.g., 0.5 to 0.95,0.5 to 0.9, 0.5 to 0.85, 0.5 to 0.8, 0.6 to 0.95, 0.6 to 0.9, 0.6 to0.85, 0.6 to 0.8, 0.7 to 0.95, 0.7 to 0.9, 0.7 to 0.85, or 0.7 to 0.8.In some embodiments, n is 0.5. In some embodiments, n is 0.55. In someembodiments, n is 0.6. In some embodiments, n is 0.65. In someembodiments, n is 0.7. In some embodiments, n is 0.75. In someembodiments, n is 0.8. In some embodiments, n is 0.85. In someembodiments, n is 0.9. In some embodiments, n is 0.95. In someembodiments, n is 1.

The term “about” refers to a range of values which can be 15%, 10%, 8%,5%, 3%, 2%, 1%, or 0.5% more or less than the specified value. Forexample, “about 10%” can be from 8.5% to 11.5%. In one embodiment, theterm “about” refers to a range of values which are 5% more or less thanthe specified value. In another embodiment, the term “about” refers to arange of values which are 2% more or less than the specified value. Inanother embodiment, the term “about” refers to a range of values whichare 1% more or less than the specified value.

The articles “a” and “an” are used in this disclosure to refer to one ormore than one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “and/or” is used in this disclosure to mean either “and” or“or” unless indicated otherwise.

The claims should not be read as limited to the described order orelements unless stated to that effect. It should be understood thatvarious changes in form and detail may be made by one of ordinary skillin the art without departing from the spirit and scope of the appendedclaims. All embodiments that come within the spirit and scope of thefollowing claims and equivalents thereto are claimed.

EXAMPLES

The disclosure is further illustrated by the following examples andsynthesis examples, which are not to be construed as limiting thisdisclosure in scope or spirit to the specific procedures hereindescribed. It is to be understood that the examples are provided toillustrate certain embodiments and that no limitation to the scope ofthe disclosure is intended thereby. It is to be further understood thatresort may be had to various other embodiments, modifications, andequivalents thereof which may suggest themselves to those skilled in theart without departing from the spirit of the present disclosure and/orscope of the appended claims.

Example 1

Described herein are illustrative examples of how these parametersindependently, as well as in combination, impact release kinetics andhow they can be leveraged to meet specific target product profiles. mAbIgG1 and mAb IgG4 are used as model monoclonal antibodies for themajority of results presented in this report with a data set alsoincluding mAb bispecific and Trap protein. In general, the resultspresented here could be extended to other monoclonal antibodies (mAbs)or proteins similar in size to the mAbs used here.

The protein hydrogel delivery system is fabricated by reacting twobranched PEG reagents with complimentary reactive end-groups in thepresence of spray dried protein suspended in an organic solvent. Uponreacting, the PEG reagents form a cross-linked network entrapping thespray dried protein, which remains in the solid state. The solvent isthen removed from the resulting mAb loaded polymer matrix, leavingbehind a solid dehydrated mAb hydrogel drug product, termed mAb-XPEG.The organic solvent is chosen for compatibility with the spray driedprotein, which needs to be insoluble in the solvent and stable, and forprocessing considerations. In all experiments presented here,dichloromethane (DCM) was used as the reaction solvent and was chosenfor its compatibility with the selected proteins as well as volatility,allowing for easy drying of the mAb-XPEG matrix at room temperatureunder vacuum. The mAb-XPEG matrix can be formed as a slab or cast as afilm or into a mold; after drying, the solid matrix can be further cutinto shapes and/or particles with specific geometries.

When the mAb-XPEG drug product is hydrated, the matrix, being ahydrogel, swells. Upon hydration, a portion of the loaded spray driedmAb will dissolve and diffuse from the matrix. In this initial phase,protein release from the matrix is diffusion based and exhibits adependence on the square root of time. The remainder of the protein,being exposed to high concentrations of PEG in the localmicroenvironments within the polymer matrix, will remain in the solidstate until hydrogel dissolution progresses to the point at which thePEG concentration in the local microenvironment drops to a levelallowing for protein dissolution. Thus, with passing time in thehydrated environment, the cross-linking in the PEG matrix degrades dueto hydrolysis, resulting in protein dissolution and subsequent diffusionfrom the polymer matrix. In this phase of release, matrix dissolution isthe rate-limiting step, and protein release is exponential as a functionof time. Finally, when the matrix has degraded to a point at which nosolid protein remains, release of the remaining protein in solutionwithin the matrix is once again controlled by diffusion through theremaining matrix. These phases of protein release from the matrix giverise to the characteristic sigmoidal shape of the release profile (FIG.1).

In Phase I of FIG. 1, upon hydration, a portion of loaded proteinexposed to surface or in regions of low PEG density is solubilized.Protein particles on or near surface are immediately released as “burst”release. This is evidenced by an increase in burst release correlatingwith an increase in surface area to volume (mass). Protein dissolvedupon hydration, presumably in regions with low PEG density, but not atsurface, is available for release in the “diffusion” phase. This isevidenced by an increase in the amount of protein released during thediffusion phase with increasing molar ratio of NH:NHS. Without wishingto be bound by theory, by increasing molar ratio, fewer cross-links areformed and regions with PEG density low enough to allow for dissolutionof protein particles will be more prevalent. In this phase, diffusionfollows Fickian diffusion with the driving force of diffusion being theconcentration of solubilized protein, but is slowed by the porousnetwork created by PEG cross-linking, and thus the rate of releaseduring this diffusion phase may be impacted by the extent and structureof the PEG cross-linking.

In Phase II of FIG. 1, the dissolution phase starts when the cleavage ofPEG cross-linking reaches an extent at which it becomes therate-limiting step for protein release from the matrix. Without wishingto be bound by theory, cleavage of PEG cross-linking is a first orderprocess, and as it occurs it is expected to both decrease PEG densitywithin the matrix, allowing for the solubilization of protein particles,as well as increase the rate of diffusion of protein through the PEGmatrix as the porous network opens up. The point in the release profilein which dissolution-controlled release is observed takes place when theoverall rate of diffusion of protein from the matrix is faster than theeffective dissolution of the cross-linking. The shape of the releaseprofile during the dissolution phase is a direct correlation to thefirst order process of dissolution. The rate of release and duration ofthe dissolution phase is expected to depend on the extent ofcross-linking (molar ratio of NH:NHS), the rate of cleavage, and thetotal amount of protein remaining in the matrix upon initiation of thisphase.

In Phase III of FIG. 1, the depletion phase occurs when once againFickian diffusion is rate limiting. In this phase, all protein haspresumably been dissolved within the matrix and the driving force ofdiffusion for release is decreasing as the remaining protein depletesfrom the matrix. This is evidenced by the characteristic shape (linearwith respect to square root of time) of the release profiles just priorto the plateau indicating depletion. Depending on protein load and thekinetics and duration of release during the diffusion and dissolutionphase, this phase may occur well before full gel dissolution.

Table 1 lists the formulation parameters theoretically associated withrelease kinetics in the protein loaded hydrogel drug delivery system.These parameters have been separated into three categories: factorsbased on the selection of polymer reagents, factors relating to thespray dried protein formulation, and factors determined in the mAb-XPEGfabrication. This list only considers formulation related parameters anddoes not include process parameters such as solvent, reagentconcentrations, reaction conditions, etc. For all studies included here,such process parameters are kept constant.

TABLE 1 mAb-XPEG Formulation Parameters Polymer Reagents Spray DriedProtein Hydrogel Fabrication (PEGNH & PEGNHS) Formulation & FormBranching (4-arm/8- Protein content Extent of cross-linking arm)Particle (“molar ratio NH:NHS”) Molecular Weight Size/Distribution SolidLoading End Group (controls Molecule Surface Area:Volume rate ofcrosslinking Powder Properties Ratio (form factor) hydrolysis)

The rationale behind focusing efforts on leveraging the protein-loadedhydrogel fabrication and spray-dried protein formulation and limitingthe choice of polymer reagents (i.e., MW and same functional end groupchemical structure) is to simplify, from a CMC (chemistry,manufacturing, and control) and regulatory perspective, the complexityof the platform by limiting the number of PEG reagents to two. However,it should be noted that similar tuning can be achieved in this system byleveraging different polymer characteristics (e.g., functional end groupchemical structure) to impact diffusion of protein within the matrix anddissolution rate of the hydrogel matrix.

Considering the spray-dried formulation parameters in understandingrelease kinetics is a key factor for platform development of thisdelivery system. While the spray-dried formulation may be leveraged toachieve desired release kinetics, the formulation may also be dictatedbased on protein stability. While molecules may behave similarly whenloaded into the hydrogel matrix in regards to release kinetics, proteinstability is known to be molecule specific and different proteins mayrequire changes to the spray-dried formulation to maintain stabilityeither in the manufacturing process or during storage as intermediatedrug substance or protein loaded hydrogels. It is therefore important,in either case, to understand how spray-dried formulations will impactrelease kinetics. This is similarly true for the surface area to volumeratio as a hydrogel fabrication parameter. While in some instancessurface area to volume may be leveraged to achieve desired releasekinetics, it may also be dictated or limited by the form factor requiredfor administration (e.g., to be syringeable or as an implant).

Materials and Methods

TABLE 2 Spray-Dried Protein Formulations, Reagents, and Materials Usedin protein-loaded hydrogel Fabrication and in vitro release (IVR)Studies Protein content in Material Spray-Dried Powder Spray Dried mAbIgG1 80% w/w Protein mAb IgG1 90% w/w Formulations mAb IgG1 50% w/w mAbIgG4 40% w/w mAb IgG4 80% w/w mAb IgG4 90% w/w mAb IgG4 50% w/w mAb 80%w/w bispecific Trap 70% w/w Protein PEG PEG-NH 8-arm NH-PEG, 14450 Da MWReagents [Hexaglycerol octa(aminopropyl)polyoxyethylene] PEG-NHS 8-armNHS-PEG, glutaryl, 45573 Da MW [Hexaglycerolocta(succinimidyloxyglutaryl)polyoxy- ethylene] Reaction DCMDichloromethane Solvent IVR Media PBS, pH 7.4 4 mM PO4, 155 mM NaCl,0.03% PS20, pH 7.4

TABLE 3 mAb-XPEG Design Space Multifactorial Study I Factor Range TestedMolar ratio 0.9-1.8 NH:NHS Solid Loading 0.6-0.9 w/w Protein Content0.4-0.8 w/w Molecule mAb IgG1, mAb IgG4

In Table 3, ranges and factors included in multifactorial analysis ofimpact of protein-loaded hydrogel formulation parameters on proteinrelease kinetics. Surface area to volume is not controlled in thisevaluation. Samples are specified in Table 4.

TABLE 4 Protein-loaded hydrogel Design Space Multifactorial Study I.Spray- Dried Total Solid Protein Actual Loading Content molar ratioSample ID Molecule (w/w) (w/w) (NH:NHS) MAB2RS001F1 mAb IgG4 0.9 0.4 1.2MAB2RS001F2 mAb IgG4 0.9 0.4 1.2 MAB2RS003F1 mAb IgG4 0.9 0.4 1.5MAB2RS005F5 mAb IgG4 0.9 0.8 1.4 MAB2RS005F6 mAb IgG4 0.9 0.8 1.6MAB2RS006F4 mAb IgG4 0.8 0.8 0.8 MAB2RS006F5 mAb IgG4 0.9 0.8 1.4MAB2RS006F6 mAb IgG4 0.9 0.8 1.6 MAB1RS004F1B mAb IgG1 0.6 0.8 1.2MAB1RS004F1C mAb IgG1 0.6 0.8 1.2 MAB1RS017F1A mAb IgG1 0.6 0.8 1.1MAB1RS017F1B mAb IgG1 0.6 0.8 1.3 MAB1RS017F1C mAb IgG1 0.7 0.8 1.8MAB1RS017F2A mAb IgG1 0.7 0.8 1.0 MAB1RS017F2B mAb IgG1 0.7 0.8 1.0MAB1RS017F2C mAb IgG1 0.7 0.8 1.0 MAB1RS017F3A mAb IgG1 0.8 0.8 1.0MAB1RS017F3B mAb IgG1 0.9 0.8 1.5

TABLE 5 Protein-loaded hydrogel Design Space Multifactorial Study II.Factor Range Tested Molar ratio 1.1-2.0 NH:NHS Solid Loading 0.2-0.7 w/wProtein Content 0.5-0.9 w/w Molecule mAb IgG1, mAb IgG4 Surface Area toVolume Ratio 3-79 (mm⁻¹)

In Table 5, ranges and factors included in multifactorial analysis ofimpact of protein-loaded hydrogel formulation parameters on mAb releasekinetics. Ranges expanded from Study I and Surface Area:Volume Ratioincluded as Factor. Samples are specified in Table 6.

TABLE 6 Protein-loaded hydrogel Design Space Multifactorial Study II.Spray- Dried Actual Total Protein molar Solid Content ratio Loading S:VS:V SampleID Molecule (w/w) (NH:NHS) (w/w) mm⁻¹ Category mAb2A mAb IgG40.5 1.1 0.7 35 High mAb2B mAb IgG4 0.9 2.0 0.7 24 High mAb2C mAb IgG40.5 1.5 0.2 79 High mAb2D mAb IgG4 0.9 1.8 0.7 6 Medium mAb2F mAb IgG40.5 2.0 0.2 3 Low mAb2G mAb IgG4 0.5 1.1 0.7 3 Low mAb2D2 mAb IgG4 0.91.8 0.7 4 Low mAb2E mAb IgG4 0.9 1.2 0.2 4 Low mAb1-A mAb IgG1 0.9 1.20.2 53 High mAb1-B mAb IgG1 0.5 1.1 0.7 4 Medium mAb1-C mAb IgG1 0.5 2.00.2 4 Medium mAb1-D mAb IgG1 0.9 1.5 0.7 4 Medium mAb1-E mAb IgG1 0.51.5 0.7 35 High mAb1-F mAb IgG1 0.5 2.0 0.2 52 High mAb1-G mAb IgG1 0.91.9 0.7 3 Low mAb1-H mAb IgG1 0.9 1.2 0.2 3 Low

The mAb-XPEG fabrication protocol is shown below.

I. Reagent Preparation:

-   -   Add PEG reagents into separate tubes.    -   Add DCM.    -   Mix thoroughly until PEG reagents are dissolved.    -   Add PEG-NH solution to each vial of spray-dried protein.    -   Swirl and sonicate to suspend protein in PEG-NH solution. And    -   Record weight after every step.

II. Crosslinking Reaction:

-   -   Add PEG-NH/Protein suspension and mix while dispensing.    -   The molar ratio (NH:NHS) is determined based on the weight of        PEG-NHS solution and PEG-NH/Protein suspension added during        fabrication assuming homogeneous solutions/suspensions.    -   Leave at room temperature in vacuum chamber or fume hood,        uncapped, overnight to allow for solvent evaporation.

Conclusions

FIGS. 2-6 show examples of the impact of independent factors on releaseprofiles.

FIGS. 7-8 shows examples of leveraging important formulation parametersto tune release profiles.

FIG. 9 shows an example of tuning factors to counteract impact of changein form factor from slab to microparticles on release kinetics.Microparticles are produced via milling and sieving and are non-uniform.The goal is to reduce burst release and achieve 45-56 days release.

Example 2

This example investigates the impact of three factors on the releaseprofile of the hydrogel: molar ratio of NH:NHS, molecular weight ofPEG-NH, and molecular weight of PEG-NHS. Molecular ratios are chosen tobe >1.0 and range 1.3-1.8 to meet target duration <60 days. Commerciallyavailable 4-arm PEG NH and NHS groups are included in the study. Themolar ratio is treated as a continuous factor, while the molecularweight of PEG-NH or PEG-NHS is treated categorically. See Table 7 fordesign parameters.

TABLE 7 Design Parameters Factor Name Role Values Molar Ratio Continuous1.3 1.8 (NH:NHS) PEG-NH MW Categorical 10 kDa 15 kDa 20 kDa 40 kDa(4-arm) PEG-NHS MW Categorical 10 kDa 15 kDa 20 kDa 40 kDa (4-arm)

In Table 7, constant parameters: 50% protein loading (mAb IgG1), 60%solid loading, and SA:V ˜23 mm⁻¹.

Table 8 below shows the different runs performed to produce thepredictive model.

TABLE 8 PEG NH PEG NHS Reagent Reagent Run # Molar Ratio (kDa) (kDa) 11.8 10 15 2 1.3 10 20 3 1.8 15 20 4 1.3 15 15 5 1.3 20 20 6 1.3 20 10 71.3 40 40 8 1.8 40 15 9 1.8 40 10 10 1.6 10 15 11 1.6 20 10 12 1.6 20 4013 1.6 40 40

FIG. 10 shows the model analysis. Power >0.95 with AC<6 indicate strongpredictive power of model.

TABLE 9 Example Parameter Tuning Capabilities Release range PEG-NHPEG-NHS Molar ratio (Time to 99% MW (kDa) MW (kDa) range Release) 10 151.3-1.8 9-31 days 40 20 1.3-1.8 22-44 days 15 10 1.3-1.8 37-59 days

As shown in Table 10 below, the accuracy of the model for the fourselected points is >99.8%. Overall accuracy of prediction equationis >99.9% in this example formulation. Prediction Accuracy (i.e.R-squared) for all fits of formulations used in the model is >98%.

TABLE 10 Prediction Model Closely Matches Experimental Data Time(days) - Time (days) - % Release measured predicted Bias (days) 23.330.9 31.3 −0.4 51.5 42.0 41.7 −0.3 72.5 45.0 45.1 −0.1 99.7 49.0 49.0 0

Table 11 summarizes the 4-arm XPEG formulations using varying molarratio, PEG NH molecular weight, and PEG NHS molecular weight withconstant protein (IgG1 mAb1), excipient loading, and surface area:volume(SA:V) ratio to achieve specified release period <60 days.

TABLE 11 Release PEG PEG Period NH MW NHS MW Molar Ratio Solid Loading(days) (kDa) (kDa) (NH:NHS) (% w/w) 9 40 15 1.83 60 14 40 15 1.71 60 2140 15 1.54 60 21 10 15 1.67 60 30 20 20 1.83 60 30 40 15 1.46 60 58 1510 1.30 60

Example 3

Table 12 shows that solvent mixture combinations and ratios can beadjusted to alter XPEG reaction time to enable scale-up manufacturing.Reaction time is defined as the time from initial mixing of solvent/PEGsolutions to time when hydrogel becomes a monolithic, solid structure.

TABLE 12 % v/v Mixture mg/mL PEG of Solvents A Reagent in Reaction TimeSolvent A Solvent B and B Solvent A/B (seconds) Methylene Ethyl 90% DCM15 95 Chloride Acetate 10% EA (DCM) (EA) Methylene Ethyl 50% DCM 15 113Chloride Acetate 50% EA (DCM) (EA) Methylene N/A 100% DCM 15 120Chloride (DCM) Methylene Chloroform 90% DCM 15 143 Chloride (Chl) 10%Chl (DCM) Methylene Chloroform 50% DCM 15 159 Chloride (Chl) 50% Chl(DCM)

EQUIVALENTS

While the present invention has been described in conjunction with thespecific embodiments set forth above, many alternatives, modificationsand other variations thereof will be apparent to those of ordinary skillin the art. All such alternatives, modifications and variations areintended to fall within the spirit and scope of the present invention.

1. A method of producing a hydrogel with a desired release profile for abiologic disposed therein, wherein the biologic is in a solid stateformulation prior to being disposed in the hydrogel, the methodcomprising: predetermining at least one of the following parameters: (a)the number of nucleophilic groups in a first precursor; (b) the numberof electrophilic groups in a second precursor; (c) the molecular weightof the first precursor; (d) the molecular weight of the secondprecursor; (e) a weight ratio of the biologic and excipient to thehydrogel; (f) a weight percentage of the biologic in the solid stateformulation; and (g) a ratio of surface area to volume of the hydrogel;determining a molar ratio of the nucleophilic group to the electrophilicgroup alone, or in combination with any one or more of the aboveparameters that is not predetermined, until the desired release profileis achieved; and crosslinking the first precursor and the secondprecursor at the determined molar ratio around the solid stateformulation under anhydrous conditions.
 2. The method of claim 1,wherein the molar ratio of the nucleophilic group to the electrophilicgroup is greater than
 1. 3. The method of claim 1, wherein the molarratio of the nucleophilic group to the electrophilic group is lessthan
 1. 4. (canceled)
 5. The method of claim 1, wherein the biologic isa recombinant protein.
 6. The method of claim 5, wherein the recombinantprotein is an antibody or a Trap protein (a fusion protein with decoyreceptor domains).
 7. The method of claim 1, wherein the nucleophilicgroup comprises a primary amine or a primary thiol.
 8. The method ofclaim 1, wherein the electrophilic group comprises succinimide,succinimide ester, n-hydroxysuccinimide, maleimide, succinate,nitrophenyl carbonate, aldehyde, vinylsulfone, azide, hydrazide,isocyanate, diisocyanate, tosyl, tresyl, or carbonyldiimidazole.
 9. Themethod of claim 1, wherein the number of the nucleophilic groups in thefirst precursor is in the range of about 2 to about
 10. 10. (canceled)11. The method of claim 1, wherein the number of the electrophilicgroups in the second precursor is in the range of about 2 to about 10.12-16. (canceled)
 17. The method of claim 1, wherein the weight ratio ofthe biologic to the hydrogel is between about 10% to about 90%.
 18. Themethod of claim 1, wherein the weight percentage of the biologic in thesolid state formulation is between about 30% to about 95%. 19-21.(canceled)
 22. The method of claim 1, wherein the desired releaseprofile comprises: (a) a release period of about two months to sixmonths for at least 90% biologic release; (b) a release period of aboutone week to two months for at least 90% biologic release; (c)near-linear release of the biologic over at least one week; (d) adelayed-release portion; (e) a sigmoidal shape; (f) a linear portion;(g) a near-linear portion; (h) a logarithmic portion; (i) an exponentialportion; or a combination thereof. 23-25. (canceled)
 26. The method ofclaim 1, wherein the crosslinking occurs in the presence of an organicsolvent that is anhydrous and hydrophobic.
 27. The method of claim 26,wherein the organic solvent is methylene chloride, ethyl acetate,dimethyl carbonate, chloroform, or a combination thereof.
 28. The methodof claim 1, wherein the determining step is performed with a predictivemodel.
 29. The method of claim 28, wherein the molar ratio has acontinuous effect on release profile in the predictive model.
 30. Themethod of claim 28, wherein the molecular weights of the first andsecond precursors have a non-continuous effect on release profile in thepredictive model.
 31. A method of producing a hydrogel having a biologicdisposed therein, wherein the biologic is in a solid state formulationprior to being disposed in the hydrogel, and wherein the hydrogel ischaracterized by a desired release period of about one week to about sixmonths for at least 90% biologic release, the method comprising:selecting a first precursor that comprises two or more nucleophilicgroups, wherein the first precursor has a molecular weight in the rangeof about 1 kDa to about 100 kDa; selecting a second precursor thatcomprises two or more electrophilic groups, wherein the second precursorhas a molecular weight in the range of about 1 kDa to about 100 kDa;determining at least one of the following parameters alone, or incombination, until the desired release period is achieved: (a) a molarratio of the nucleophilic group to the electrophilic group; (b) a weightratio of the biologic and excipient to the hydrogel; (c) a weightpercentage of the biologic in the solid state formulation; and (d) aratio of surface area to volume of the hydrogel; and crosslinking thefirst precursor and the second precursor at the determined molar ratioaround the solid state formulation under anhydrous conditions.
 32. Themethod of claim 31, wherein the molar ratio of the nucleophilic group tothe electrophilic group is greater than
 1. 33. The method of claim 31,wherein the molar ratio of the nucleophilic group to the electrophilicgroup is less than
 1. 34. (canceled)
 35. The method of claim 31, whereinthe biologic is a recombinant protein.
 36. The method of claim 35,wherein the recombinant protein is an antibody or a Trap protein (afusion protein with decoy receptor domains).
 37. The method of claim 31,wherein the nucleophilic group comprises a primary amine or a primarythiol.
 38. The method of claim 31, wherein the electrophilic groupcomprises succinimide, succinimide ester, n-hydroxysuccinimide,maleimide, succinate, nitrophenyl carbonate, aldehyde, vinylsulfone,azide, hydrazide, isocyanate, diisocyanate, tosyl, tresyl, orcarbonyldiimidazole.
 39. The method of claim 31, wherein the firstprecursor comprises about 2 to about 10 nucleophilic groups. 40.(canceled)
 41. The method of claim 31, wherein the second precursorcomprises about 2 to about 10 electrophilic groups. 42-44. (canceled)45. The method of claim 31, wherein the weight ratio of the biologic tohydrogel is between about 10% to about 90%.
 46. The method of claim 31,wherein the weight percentage of the biologic in the solid stateformulation is between about 30% to about 95%. 47-49. (canceled)
 50. Themethod of claim 31, wherein the desired release period comprises: (a) arelease period of about two months to six months for at least 90%biologic release; (b) a release period of about one week to two monthsfor at least 90% biologic release; (c) near-linear release of thebiologic; (d) a delayed-release portion; (e) a sigmoidal shape; (f) alinear portion; (g) a near-linear portion; (h) a logarithmic portion;(i) an exponential portion; or a combination thereof. 51-53. (canceled)54. The method of claim 31, wherein the crosslinking occurs in thepresence of an organic solvent that is anhydrous and hydrophobic. 55.The method of claim 54, wherein the organic solvent is methylenechloride, ethyl acetate, dimethyl carbonate, chloroform, or acombination thereof.
 56. The method of claim 31, wherein the determiningstep is performed with a predictive model.
 57. The method of claim 56,wherein the molar ratio has a continuous effect on release period in thepredictive model.
 58. The method of claim 56, wherein the molecularweights of the first and second precursors have a non-continuous effecton release period in the predictive model.
 59. A hydrogel comprising abiologic produced by the method of claim 1.